PROJECT SUMMARY Eukaryotic DNA is packaged with histone proteins to form nucleosomes, which in turn condense into higher-ordered structures that constitute the different functional forms of chromatin. It is well accepted that chromatin structural states such as heterochromatin (highly condensed/ transcriptionally inactive) or euchromatin (decondensed/ transcriptionally active), can be propagated in an epigenetic fashion (i.e. associated with heritable changes in phenotype that are not related to changes in DNA sequence). Importantly, mis-regulation of chromatin structure and posttranslational modifications on histones are linked to cancer and developmental diseases. Characterizing the molecular mechanisms regulating these epigenetic ?on?/ ?off? pathways requires identification of new histone modification states, and development of new methodologies that permit comprehensive studies and unbiased screens of factors bound to distinct chromatin regions. To this end, we recently found a new histone modification, H3K23me3, using a specialized, entirely heterochromatic nucleus in the model organism Tetrahymena thermophila, and characterized it as a ?mark? of the pericentric chromatin that is important for maintaining genome integrity during meiosis. In this proposal, our goal is to follow up on our initial discovery by studying the enzymology associated with catalyzing and removing H3K23me3, as well as characterizing the biological role of this heterochromatin marker in humans and other mammals, where it may also be involved in neurogenesis. It is also worthwhile to note that we will comprehensively analyze the epiproteome of H3K23me3-associated chromatin in mammals using improvements on our new technology termed rCRISPR-ChAP-MS, which provides for the analysis of macromolecular protein interactions on chromatin at a defined genomic position in vivo. In doing so we will test the hypothesis that H3K23me3, through interactions with H3K23me3 binding proteins, helps to pinpoint, protect, and perpetuate sites of heterochromatin formation during meiosis and cell differentiation. To test our hypothesis and work towards our short term goal, we will pursue the following three Aims: (1) Characterize the enzymology and biological impact of H3K23me3 in Tetrahymena, (2) Characterize the role of H3K23me3 in mammalian meiosis, and (3) Characterize the role of H3K23me3 in mammalian neuronal development. Our study of the molecular underpinnings of how heterochromatic histone PTMs like H3K23me3 contribute to epigenetic silencing should help address our long term goal of understanding transgenerational inheritance of epigenetic modifiers, and may introduce therapeutic targets for human diseases associated with disrupted gene silencing or heterochromatin pathways.